Processes for the conjugation of the double bonds of polyunsaturated unconjugated fatty acids have found their main application in the field of paints and varnishes. Oils comprised of triglycerides of conjugated fatty acids are known as drying oils. Drying oils have value because of their ability to polymerize or “dry” after they have been applied to a surface to form tough, adherent and abrasion resistant films. Tung oil is an example of a naturally occurring oil containing significant levels of fully conjugated fatty acids. Because tung oil is expensive for many industrial applications, research was directed towards finding substitutes.
In the 1930's, it was found that conjugated fatty acids were present in oil products subjected to prolonged saponification, as originally described by Moore, J. Biochem., 31: 142 (1937). This finding led to the development of several alkali isomerization processes for the production of conjugated fatty acids from various sources of polyunsaturated fatty acids.
The positioning of the double bonds in the hydrocarbon chain is typically not in a conjugated, i.e., alternating double bond single bond double bond, manner. For example, α-linolenic acid is an eighteen carbon acid with three double bonds (18:3) at carbons 9, 12 and 15 in which all three double bonds have in the cis configuration, i.e., 9Z,12Z,15Z. γ-Linolenic acid is 6Z,9Z,12Z—C18:3 acid.
Migration of double bonds (e.g., leading to conjugation) gives rise to many positional and geometric (i.e., cis-trans) isomers.
Conjugated double bonds means two or more double bonds which alternate in an unsaturated compound as in 1,3 butadiene. The hydrogen atoms are on the same side of the molecule in the case of cis structure. The hydrogen atoms are on opposite sides of the molecule in the case of trans structure.
Conjugated linoleic acid (CLA) is a general term used to name positional and geometric isomers of linoleic acid. Linoleic acid is a straight chain carboxylic acid having double bonds between the carbons 9 and 10, and between carbons 12 and 13. For example, one CLA positional isomer has double bonds between carbons 9 and 10 and carbons 11 and 12 (i.e, 9Z, 11E-C18:2 acid); another has double bonds between carbons 10 and 11 and carbons 12 and 13 (i.e., 10E,12Z—C18:2 acid), each with several possible cis and trans isomers as shown in the following Table:
TABLENuFatty AcidTrivial NameStructure19Z, 12Z, 15Z-C18:3α-Linolenic Acid 26Z, 9Z, 12Z-C18:3γ-Linolenic Acid 39Z, 12Z-C18:2Linoleic Acid
Conjugated linolenic acid (CLNA) is a general term used to name positional and geometric isomers of linolenic acid. Linolenic acid is a straight chain carboxylic acid having double bonds between the carbons 9 and 10, between the carbons 12 and 13 and between carbons 15 and 16 (see the above Table).
The 9Z,11E-C18:2 isomer has been shown to be the first intermediate produced in the biohydrogenation process of linoleic acid by the anaerobic rumen bacterium Butyrvibrio fibrisolvens. This reaction is catalyzed by the enzyme Δ11 isomerase which converts the cis-12 double bond of linoleic acid into a trans-11 double bond. (C. R. Kepler et al., 241 J. Biol. Chem. (1966) 1350). It has also been found that the normal intestinal flora of rats can also convert linoleic acid to the 9Z, 11E-C18:2 acid isomer. The reaction does not, however, take place in animals lacking the required bacteria. Therefore, CLA is largely a product of microbial metabolism in the digestive tract of primarily ruminants, but to a lesser extent in other mammals and birds.
Conjugated Linoleic and Linolenic Acids in Cancer Therapy
The free, naturally occurring conjugated linoleic acids (CLA) have been previously isolated from fried meats and described as anticarcinogens by Y. L Ha, N K. Grimm and M. W. Pariza, in Carcinogenesis, Vol. 8, No. 12, pp. 1881-1887 (1987). Since then, they have been found in some processed cheese products (Y. L. Ha, N. K. Grimm and M. W. Pariza, in J. Agric. Food Chem., Vol. 37, No. 1, pp. 75-81 (1987)).
Conjugated Linolenic Acid (CLNA) is naturally present as a minor component of cheese from cow milk (Winkler et al., 2001) and bovine milk fat (Destaillats et al., 2003).
Cancer is a complex multifactor and multistep process involving the coordinated expression and suppression of genes functioning as positive and negative regulators of oncogenesis (Fisher, 1984; Bishop, 1991; Knudson et al., 1991; MacLachlan et al, 1995). Solid tumors are the leading cause of death attributable to cancers worldwide. Conventional methods of treating cancer include surgical treatments and the administration of chemotherapeutic agents. However, to date, such treatments have been of limited success. Chemotherapeutic treatments available today are also of limited usefulness because of their non-selective killing and/or toxicity to most cell types. Also, many tumor cells eventually become resistant against the chemotherapeutic agent, thus making treatment of solid tumors and other tumors non-feasible.
Cells can die either from apoptosis or necrosis. Unlike necrosis which is a pathological cell death, apoptosis is a death which is initially programmed in the gene of the cell itself. Thus, the gene which programs the apoptosis is activated by certain external or internal causes whereby programmed cell death gene protein is produced based upon said gene and then the cell itself is decomposed and dead by the resulting programmed death protein. Cells that undergo apoptotic cell death are characterized by a number of functional and morphologic changes: loss of membrane asymmetry, which results in the exposure of phosphatidylserine (PS) on the outer surface of cell membrane; loss of the inner mitochondrial membrane potential; activation of cytoplasmic serine proteases (caspases); rapid formation of extrusions of the cell membrane, which results in the formation of small extracellular membrane-coated particles (bleds); shrinkage of the total cell volume; condensation of the nuclear chromatin, which leads to the shrinkage of the nucleus, and fragmentation of the nucleus and the remaining cytoplasm into apoptotic bodies (Cohen, 1993).
Anti-carcinogenic properties of CLA have been well documented, as well as stimulation of the immune system. Administration of CLA inhibits rat mammary tumorogenesis, as demonstrated by Ha et al., Cancer Res., 52:2035-s (1992). Ha et al., Cancer Res., 50:1097 (1990), reported similar results in a mouse forestomach neoplasia model. CLA has also been identified as a strong cytotoxic agent against target human melanoma, colorectal and breast cancer cells in vitro. A recent major review article confirms the conclusions drawn from individual studies (Ip, Am. J. Clin. Nutr. 66(6):1523s (1997)). In in vitro tests, CLAs were tested for their effectiveness against the growth of malignant human melanomas, colon and breast cancer cells. In the culture media, there was a significant reduction in the growth of cancer cells treated with CLAs by comparison with control cultures. The mechanism by which CLAs exert anticarcinogenic activity is unknown.
In addition, CLAs have a strong antioxidative effect so that, for example, peroxidation of lipids can be inhibited (Atherosclerosis 108, 19-25 (1994)). CLA has been found to be an in vitro antioxidant, and in cells, it protects membranes from oxidative attack. In relation to other important dietary antioxidants, it quenches singlet oxygen less effectively than beta-carotene but more effectively than alpha-tocopherol. It appears to act as a chain terminating antioxidant by chain-propagating free radicals (U.S. Pat. No. 6,316,645).
Pharmaceuticals which have been used in clinical therapy include many agents such as anticancer agents, antibiotic substances, immunopotentiators, immunomodulators, etc. (such as alkylating agents antimetabolites and plant alkaloids) but it can be hardly said that such a drug therapy has been completely established already. An object of the present invention is to develop a substance having a physiological function such as apoptosis-inducing action.
Conjugated linoleic acid (CLA) is a general term used to name positional and geometric isomers of linoleic acid C18:2(9 cis,12 cis). It usually denotes a mixture of mainly two isomers: C18:2(9cis, 11trans) and C18:2(10trans,12cis). They are usually present in a 1:1 ratio and the sum of these two isomers can vary between 30% and 90%. The majority of CLA in nutraceutical market do not mention the accurate composition for the content of each isomer, but generally the product is around 80% for both isomers. The most important isomer in term of anti-cancer activity is the C18:2(9cis, 11trans) (Seidel et al, 2001, U.S. Pat. No. 6,319,950, Liu et al., 2002, Roche et al., 2002, Pariza et al, 1991).
CLA have been suggested as useful as anti-cancer agents for treatment of cancer. The latest research reveals the most dramatic impact may be on the reduced risk and incidence of mammalian cancer (breast and colon cancer). It has been shown that CLA down-regulated mammary growth, decrease the population and proliferation activity of the cancer cells, and therefore reduces mammary cancer risk and metastasis in mice and rats (Ha et al, 1987, Ip et al, 1999). The growth inhibitory effect of CLA was also demonstrated on human breast cancer cells (Durgam et al., 1997).
Horrobin et al., in U.S. Pat. No. 6,245,811 disclosed a method for treating a disorder like complications of cancer; with compounds of structure containing group like CLA, as fatty esters as bioactive compounds
Seidel et al., in U.S. Pat. No. 6,319,950 disclosed a method for the treatment of carcinoma in a human, including administering to a human a therapeutically effective amount of C18 (9-cis, 11-trans). This patent includes administering to a human a purified conjugated linoleic acid (CLA) produced by a novel synthesis process for producing C18 (9-cis, 11-trans).
Das et al., in U.S. Pat. No. 6,426,367 disclose methods of selectively reducing the blood supply to a neoplastic region, such as a tumor region, thereby selectively causing necrosis of the neoplastic tissue without substantial necrosis of adjoining tissues. The methods described in this patent employ intra-arterial injection of polyunsaturated fatty acids, such as CLA, preferably in the form of salts, preferably with a lymphographic agent, and optionally with an anti-cancer drug, and/or a cytokine.
Das et al., in U.S. Patent No. US2002077317 disclosed a method of stabilizing and potentiating the actions of 2-methoxyoestradiol, statins, H2 blockers, and C-peptide of proinsulin which have modified influence on angiogenesis and inhibiting the growth of tumor cells, as applicable by using in coupling conjugation certain polyunsaturated fatty acids (PUFAs) chosen from linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, cis-parinaric acid or conjugated linoleic acid in predetermined quantities.
Bin et al in Patent No. CN1371985 disclosed a health-care wine containing conjugated linoleic acid or conjugated linoleic acid derivative. Said wine not only has the features of general drinking wine, but also possesses the health-care functions of resisting cancer, resisting atherosclerosis, regulating and controlling metabolism, raising immunity, regulating blood sugar and promoting growth development.
Bin et al, in Patent No. CN1356386 disclosed a process for preparing conjugated linoleic acid from dewatered castor oil includes physicochemically induced isomerizing, hydrolysis and multi-step separation. The resultant product contains conjugated linoleic acid (higher than 80%), linoleic acid (higher than 15%) and their isomers. It features its functions of preventing and treating cancer, diabetes and atherosclerosis, improving immunity, reducing blood sugar and fat.
Focant et al., in Patent No. WO02051255 relates to methods for altering the fatty acid composition in milk or tissue fat directly derived from a milk producing ruminant. In this patent methods are disclosed to obtain said desirable fatty acid profile, thereby improving the nutritional benefits to human health associated with CLA. Dietary intakes of CLA [C18:2 cis-9, trans-11] and C18:1 trans-11 fatty acids in milk or meat, or products thereof, produced in accordance with this invention in ruminant animals, can be effective in preventing cancer in different sites, reduce risk of coronary heart disease and to enhance immune function.
U.S. Pat. No. 5,554,646 (Cook et al.) discloses animal feeds containing CLA, or its non-toxic derivatives, e.g., such as sodium and potassium salts of CLA, as an additive in combination with conventional animal feeds or human foods. CLA makes for leaner animal mass.
The biological activity associated with CLAs is diverse and complex (Pariza et al. in Prog. Lipid Research., Vol 40, pp. 283-298).
Conjugated trienoic fatty acids have been suggested as useful compounds in the treatment of cell growth. Cytotoxic and anticarcinogenic effects of conjugated trienoic fatty acids have been shown on rat mammary carcinogenesis model (Futakuchi et al., 2002, Tomoyuki et al., in Patent No. JP2000336029). Same effects were observed on some lines of human tumor cells, possibly due to the induction of apoptosis of the cells (Igarashi et al., 2000a,b). In all of these studies, the authors demonstrated some properties of conjugated trienoic fatty acids, but the structure, the geometrical and positional isomers of conjugated trienoic fatty acids responsible for these effects remain to be elucidated. CLnA™ may provide potent new therapeutic molecules for the treatment of disorders such as cancers.
Tomoyuki et al, in Patent No. JP2000336029 relates to a new inhibiting agent useful in food and medicinal fields by incorporating a conjugated linolenic acid. This breast cancer-inhibiting agent contains a conjugated linolenic acid (e.g. 9,11,13-octadecatrienic acid, 10,12,14 octadecatrienic acid, their mixtures.). The breast cancer-inhibiting agent can be used not only as a medicine but also as a breast cancer-inhibiting or preventing food (e.g. a conjugated linolenic acid-containing oil and fat product), and in both cases of usage, the conjugated linolenic acid to be ingested is generally 0.01-3%, preferably 0.05-1% of the food weight.
The resemblance between the most important isomer of CLA [C18:2(9cis, 11trans)] and one of the isomers of CLnA™ [C18:3(9cis,11trans,15cis)] in term of their structure is the 9cis, 11trans insaturation. We can say that this isomer has a “CLA characteristic”. The major difference between both isomers is the third insaturation: 15cis. This insaturation confers a “omega-3 fatty acid characteristic”. This should increase the bioavaibility of the product and therefore increase the activity of CLnA™. The aims of the current studies are intended to demonstrate the additive effects of these two characteristics (CLA and omega-3 fatty acid in the same molecule).
Process of Preparation of Conjugated Linoleic or Linolenic Acids
All the useful methodologies for preparation of conjugated linoleic acid (CLA) were recently reviewed by Adlof (In:Yurawecz et al. (Ed), Advances in Conjugated Linoleic Acid Research, volume 1, AOCS Press, Champaign, II, pp 21-38 [1999]).
The usual methodology for conjugation of polyunsaturated fatty acids is alkali-catalyzed isomerization. This reaction may be performed using different bases such as hydroxides or alkoxides in solution in appropriate alcoholic reagents. This reaction was developed in the 1950's for spectrophotometric estimation of polyunsaturated fatty acids in fats and oils [AOCS official method Cd 7-58; JAOCS 30:352 (1953)].
In alkali isomerization the fatty acids are exposed to heat, pressure and a metal hydroxide or oxide in nonaqueous or aqueous environments, resulting in the, formation of conjugated isomers. Other methods have been described which utilize metal catalysts, which is not as efficient in the production of conjugated double bonds. It was found that isomerization could be achieved more rapidly in the presence of higher molecular weight solvent.
Kass, et al., J. Am. Chem. Soc., 61: 4829 (1939) and U.S. Pat. No. 2,487,890 (1950) showed that replacement of ethanol with ethylene glycol resulted in both an increase in conjugation in less time.
U.S. Pat. No. 2,350,583 and British Patent No. 558,881 (1944) achieved conjugation by reacting fatty acid soaps of an oil with an excess of aqueous alkali at 200-230 degrees Celsius and increased pressure.
Dehydration of methyl ricinoleate (methyl 12-hydroxy-cis-9-octadecenoate) (Gunstone and Said, Chem. Phys. Lipids 7, 121 [1971]; Berdeaux et al., JAOCS 74, 1011 [1997] give 9Z,11E-C18:2 isomer as a major product. U.S. Pat. No. 5,898,074 disclosed a synthesis process for producing this fatty acid at room temperature in high yield. The tosylate or the mesylate of the methyl ester of ricinoleic acid is formed with tosyl chloride or mesyl chloride in a pyridine solvent or in acetonitrile and triethyl amine. The obtained tosylate or mesylate is reacted with diazabicyclo-undecene in a polar, non-hydoxylic solvent of acetonitrile to form the preferred isomer of 9c,11t-18:2 methyl ester in high yield.
U.S. Pat. No. 6,160,141 disclosed a synthesis process for producing conjugated eicosanoid fatty acid from methyl lesquerolate (methyl 14-hydroxy-cis-11-octadecenoate) at room temperature in high yield using the same principle.
Among the processes known to effect isomerization without utilizing an aqueous alkali system, is a nickel-carbon catalytic method, as described by Radlove, et al, Ind. Eng. Chem. 38: 997 (1946). A variation of this method utilizes platinum or palladium-carbon as catalysts. Conjugated acids may also be obtained from a-hydroxy allylic unsaturated fatty acid using acid catalyzed reduction (Yurawecz et al., JAOCS 70, 1093 [1993]), and partial hydrogenation of conjugated acetylenic acid such as santalbic (11E-octadec-9-ynoic) acid using Lindlar's catalyst could also be used but are limited by natural sources of such fatty acid. Another approach uses strong organic bases such as butyllithium It has been applied to both the conjugation of linoleic acid and partial and full conjugation of alpha-linolenic acid ((U.S. Pat. No. 6,316,645 (Sih, et a)).
Main difference between all these procedures and the present invention is the fact that linolenic acid has three double bounds (9cis, 12cis, 15cis) that are much more reactive than the two double bonds of linoleic acid (9cis, 12cis). More precisely, the octatrienoic system (C18:3) is responsible for a sigmatropic rearrangement (see FIG. 1) that conduces to the formation of cyclic compounds (C18:3 11,13 cyclohexadiene) that are not possible to be formed during the isomerisation of the octadienoic system (C18:2). A rigorous control of the reaction kinetic's was necessary to maximize the yield of the desire mixture of isomers and minimize the amount of cyclic compounds. In fact, purification steps used in this invention are set in order to separate these cyclic compounds.
In the development of commercial compounds of linolenic acids known under the trademark CLnA™ it is important to have an inexpensive process to produce specific compositions that could be used in different formulations like nutritional bars and beverages, yoghurts, ice creams, cheese, butter, etc.
Natural fully conjugated linolenic acids have been found at high content levels in some seed oils (Hopkins, In:Gunstone, F. D. (Ed), Topics in Lipid Chemistry, volume 3, ELEK Science, London, pp 37-87 [1972]). For example, Takagi and Itabashi (Lipids 16, 546 [1981]) reported calendic acid (8E,10E,12Z—C18:3 acid, 62.2%) in pot marigold seed oil, punicic acid (9Z,11E,13Z—C18:3 acid, 83.0%) in pomegranate seed oil, α-eleostearic acid (9Z,11E,13E-C18:3 acid) in tung (67.7%) and bitter gourd (56.2%) seed oils, and catalpic acid (9E,11E,13Z—C18:3 acid, 42.3%) in catalpa seed oil, respectively.
An octadecatrienoic acid isomer whose structure has been tentatively defined as 9Z,11E,15Z—C18:3 acid, is believed to be the first intermediate in the biohydrogenation process of α-linolenic acid by the anaerobic rumen bacterium Butyrvibrio fibrisolvens (C. R. Kepler and S. B. Tove 242 J. Biol. Chem. (1967) 5686).
There is thus a need to provide a process for producing at a lower cost and at a high yield conjugated linolenic acid.
There is also a need to find new conjugated fatty acids that may be easily obtained through a process for its use and the treatment of cancer.